Active proton secretory transport mechanisms establish and maintain a luminal acidic environment in the excurrent ducts of the male reproductive tract. The low luminal pH and a low bicarbonate concentration are important for sperm maturation, the prevention of premature activation of acrosomal enzymes, and for maintaining sperm in a quiescent state during storage in this organ. Despite this critical function in male reproductive physiology, acidification processes in the male reproductive tract are still incompletely understood. A novel physiological approach using the non-invasive self-referencing proton-selective electrode, in collaboration with Peter Smith, had allowed us to identify the major proton secretory mechanism in the initial part of the vas deferens. We have shown that a bafilomycin-sensitive, proton-pumping H+-ATPase (proton pump - PP) is concentrated in the apical membrane of a subpopulation of epithelial cells (PP-rich cells) and is a major contributor to luminal acidification (1,2,3). We are now further characterizing proton secretion in the male reproductive tract in order to understand how defective acidification may impair reproductive function in pathologic states, and how intervention to manipulate acidification may eventually be used to control male fertility. Our specific aims are: 1) to define and characterize the complete pathways of proton secretion by the PP-rich cells; 2) to elucidate the mechanisms of vesicle recycling underlying the regulation of proton secretion; and 3) to examine the effect of some environment pollutants on proton secretion. Proton secretion in the male reproductive tract: involvement of Cl--independent HCO3- transport: Am. J. Physiol. 275 (Cell Physiol. 44): in press The lumen of the epididymis is the site where spermatozoa undergo their final maturation and acquire the capacity to become motile. An acidic luminal fluid is required for the maintenance of sperm quiescence and for the prevention of premature activation of acrosomal enzymes during their storage in the cauda epididymis and vas deferens. We have previously demonstrated that a vacuolar H+ATPase (PP) is present in the apical pole of apical and narrow cells in the caput epididymis and clear cells in the corpus and cauda epididymis, and that this PP is responsible for the majority of proton secretion in the proximal vas deferens. We now show that PP-rich cells in the vas deferens express a high level of carbonic anhydrase II (CAII), and that acetazolamide markedly inhibits the rate of proton secretion by 46.2 1 6.1%. The rate of acidification was independent of chloride and was strongly inhibited by SITS under both normal and chloride-free conditions (50.6 1 5.0% and 57.5 1 6.0%, respective ly).In the presence of chloride, diphenylamine-2-carboxylate (DPC) had no effect, whereas SITS inhibited proton secretion by 63.7 1 11.3% when applied together with DPC. In chloride-free solution, DPC markedly inhibited proton efflux by 45.1 1 7.6%, SITS produced an additional inhibition of 18.2 % 1 6.6%, and bafilomycin had no additive effect. In conclusion, we propose that CAII plays a major role in proton secretion by the proximal vas deferens. Acidification does not require the presence of chloride, but DPC-sensitive chloride channels might contribute to basolateral extrusion of HCO3- under chloride-free conditions. The inhibition by SITS observed under both normal and Cl-free conditions indicates that a Cl-/HCO3- exchanger is not involved and that an alternative HCO3- transporter participates in proton secretion in the proximal vas deferens.